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PART IV, continued:

  Among the soil microorganisms there are forms that inhibit the growth of other microbes. They are usually called antagonists.

  There is no fundamental difference between inhibitors and antagonists. Both groups act with the aid of special metabolites in their metabolic products: the inhibitors affect cells of higher organisms and the antagonists act on lower organisms. Even this distinction is not always sharp, since there are inhibitors, which also suppress microbes and, on the other hand, there are antagonists that are also toxic to plants.

  As was already mentioned, substances formed by inhibitors are called toxins or phytotoxins, and substances produced by antagonists are called antibiotics. This differentiation is of a purely formal or conventional nature. As is well known, there are many substances among the antibiotics which are highly toxic to plants and animals.

  Regardless of this relativity of concepts and designations the antibiotics and their producers are a special branch of science and are considered as special substances with specific manifestations.

  Microbial antagonism has caught the attention of scientists for many years. Pasteur, Mechnikov and their contemporaries noticed the ability of some species of microbes to suppress the growth of others (cf. Nakhimovskaya, 1937; Waksman, 1947; Krasil'nikov, 1950 and others). Pasteur observed this ability in the anthrax bacillus in relation to the microbe causing chicken cholera. Mechnikov found it in the lactobacilli in relation to the putrefactive bacteria and certain colon-type bacilli. On this basis he devised a method of changing the intestinal flora and sanitation of the human and animal intestines. The phenomenon of antagonism was observed in various groups of microorganisms, among bacteria, fungi, actinomycetes, algae, protozoa, etc. Microbial antagonists acting upon various pathogenic bacteria against cocci (staphylococci, streptococci, pneumococci and diplococci), against organisms causing intestinal infections (dysentery, parathyphoid, typhoid and cholera) against the tubercle bacillus, diphtheria, peat and anthrax brucellosis, tularemia and gas gangrene have been described. A great number of antagonists were described acting against pathogenic fungi, yeasts, protozoa, etc.

  Recently, the efforts of investigation have been concentrated on the detection of antagonists acting against viruses and malignant tumors, Antagonists of viruses and tumors can be found among actinomycetes and bacteria (Waksman, 1953; Kashkin, 1952; Kurylowicz and Slopek, 1955).

  Much data are available on the suppressing action of antagonists of phytopathogenic bacteria, actinomycetes and fungi. Antagonists were found acting against various organisms. The greatest attention is paid to antagonists--actinomycetes.

  Among 20 cultures of actinomycetes isolated from rhizosphere soil, Lochhead and Lauderkin (1949) found 11 cultures that suppressed the growth of phytophathogenic strains of Actinomyces scabies.

  Meredith and Semeniuk (1945-47) found that among the actinomycetes which they studied 21% inhibited growth of the fungus Pythium graminicola, which causes root necrosis in a number of plants. The actinomycete-antagonists of Chalaria quercina which cause wilt in the oak, and actinomycete-antagonists of Cerasto mella ulni, which affect woody plants, such as the elm, etc have been described (Stallings, 1954).

  From the soils of the southern shore of Crimea, Petrusheva (1953) isolated 31 cultures of actinomycetes. Twenty-two of these inhibited the growth of the fungi Thielaviopsis basicola which causes root rot of tobacco plants, and Fusarium sp. which causes the "black foot" disease of citrus saplings, Leben and Keitt ( 1948) had a collection of actinomycetes that inhibited 33 species of phytopathogenic fungi.

  Cooper and Chilton (1950) have done much work on the detection of actinomycete-antagonists of the phytopathogenic fungus Pythium arrhenomonas which is widely distributed in the soils of Louisiana. Out of 8,302 strains which they isolated, 18.5 to 31.5% were antagonists. Kublanovskaya (1950) noted actinomycete -antagonists to the agent of wilt of the cotton plant-- Verticillium dahliae and Fusarium vasinfectum. Lechevalier et al., (19 5 3) tested 197 strains of actinomycetes for antagonism to Cerastomella ulni and found only one active strain. Actinomycete-antagonists were found in soils which were active against phytopathogenic fungi--Helminthosporium sativum, H. victoriae, Coletotricum circinans, Verticillium albo-atrum and others (Stevenson, 1954; Stessel et al., 1953).

  In our studies we found actinomycetes in soil, which suppress phytopathogenic fungi., Fusarium lin, F. solani, F. vasinfectum, Helinthosporium sativum, Alternaria humicola, Rhizoctonia solani, Botrytis alii, Deuterophoma tracheiphilus, Trichoderma lignorum, Monila fructigena, and also fungi of the genera Penicillium, Aspergillus, Cladosporium, Verticillium and others.

  Among fungi there are many antagonists. Antagonists have been described against agents of various diseases: Fusarium, Peziza, Rhizoctonia, Ophiobolus, Botrytis, Monilia, Sporotrichum, Pythium, Phymatotricum, Phytophthora and Sclerotium.

  Porter (1924) described antagonistic relations between fungi and bacterial antagonists, and the phytopathogenic fungi Helminthosporium and Fusarium. Sanford and Broadfoot (1931) isolated from the soil 6 species of fungi which inhibit the growth and activity of the fungus Ophiobolus graminis. Weindling (1932, 1948) described a case of parasitism of the fungus Trichoderma lignorum on the fungi of the genus Rhizoctonia and others. The same was observed by Novogrudsk (1936). The latter given a long list of fungi and bacterial antagonists, indicating the species of fungi and bacteria inhibited by them.

  In this list appear more than 30 fungal species which inhibit over 50 fungi belonging to different genera and families. Many other investigators also found antagonists among the fungi (Allen and Haenseler, 1935; Joseph, 1952; Vasudeva, 1950, 1953 and others).

  Stemmel, Leben and Keitt (1953) isolated 170 fungi antagonistic to other fungi from soils.

  Anwar (1949) found that among soil fungi and bacteria approximately half (out of 86 studied) suppressed the growth of the fungus Helminthosporium sativum, and about 12% inhibited Fusarium lini. From various soils, Gregory at al., (1952) isolated 14 cultures of fungi, 29 strains of actinomycetes and 31 strains of bacteria, which actively suppressed the growth of the fungus Pythium debaryanum. Three actinomycete strains and 1 bacterial strain suppressed growth of the root-nodule bacteria, Rh. meliloti and Rh. trifolii.

  Among the sporeforming and nonsporeforming bacteria, it is not uncommon to encounter antagonists to phytopathogenic microbes. Among the sporeformers antagonists to various phytopathogenic fungi have been described. Porter (1924) mentions a number of bacteria which inhibit the growth of Helminthosporium. The latter did not grow in the presence of Bac. capoulatus, Bac. mesentericus and Bac. mycoides. Bamberg (1931) indicated inhibition of growth of the fungi Tilletia tritici, Ustilago zeae, U. levis, U. avenae by the sporeforming bacillus Bacillus D.

  The afore-mentioned bacteria showed similar action against phytopathogenic fungi such as Penicillium sp., Helminthosporium, Ophiobolus, Acrostalagmus, Fusarium. Sclerotinia gleosporium, Alternaria and others (Novogrudskii, 1936; Weindling, 1946; Stessel et al., 1953; Stallings, 1954; Krasil'nikov, 1953 a and others).

  Many antagonists have been described among the nonsporeforming bacteria. Studies show that they are most frequently encountered among the Pseudomonas and Bacterium species and also among myxobacteria. Bisby (1919) described the species Pseudomonas phaseoli which inhibited the fungus Fusarium oxysporium. Fawcett (1931) indicated Ps. juglandis as an antagonist of the fungus Dothlorella gregaris. Johnson and Marvin (1931) detected the antagonistic action of certain nonsporeforming bacteria (Bacterium C-1. Bacterium X, etc) on growth of the fungi Ustilago zeae, U. avenae, Alternaria solani, A. brassicae and A. Tenuis.

  Khudiyakov (1935) isolated and studied in detail bacteria which dissolve the mycelium of the fungi Fusarium graminearum, F. culmorum, F. scirpi, F. lini. F. herbarum, F. equiseti, Sclerotinia libertiana and others. These bacteria were called mycolytic bacteria. Subsequently, many other investigators detected these bacteria in the soil ( Raznitsyna, 1942; Berezova, 1932; Korenyako, 1939; Kublanovskaya, 1953, etc).

  Ark and Hunt (1941) had cultures of the bacteria Bac. vulgatus and Bac. sp. that inhibited the growth of many phytopathogenic bacteria--Bact. amylovorum Bact. aroideae, Bact. carotovorum, Bact. phytophthorum, Ps. campestris, Ps. lachrymans and others. These bacteria also inhibited certain fungi--Fusarium graminearum, F. lycopersici, Phytophthora sp. and others. Similar data are given by other authors (Johnson, 1935, Weindling, 1946; Christensen and, Davis, 1940; Vasudea, 1952 and Skinner, 1956).

  Antagonists to phytopathogenic fungi have been described among the myxobacteria (Johnson and Marvin, 1931; Kononenko, 1937).

  Fungi which attack nematodes are described in the literature. They were first described by M. S. Voronin in his work "Mycological Studies" published in 1869 and by Sorokin in 1871. In a series of papers Soprunov (1954) showed that these fungi differ in their species composition and are very widespread in soils. The majority of them belong to the Hyphomycete, to the genera Trichothecium, Arthrobotrys, Dactylaria, Dactylella, etc.

  These fungi "catch" the nematode with their hyphae and poison it with their metabolites. Attempts were made to use these fungi in the struggle against phytopathogenic nematodes. The introduction of this fungus into the soil lowers the incidence of plant disease. In the struggle with nematodes which affect cucumbers, the fungi-antagonists, or an they are called the predatory fungi noticeably decrease the incidence of disease; in the control group there were 23 galls per each plant and in the treated plants, an average of 0.6 galls (Soprunov, 1954).

  According to Gorlenko (1955), artificial enrichment of soil by predatory fungi lowers the morbidity of cucumbers 1.5-7 times. Tendetnik (1957) used the predatory fungi for exterminating pathogenic larvae, the ancylostomes in mines and also to destroy strongyles in the manure of infected animals.

  On the basis of existing evidence, one may say that there are no species of bacteria. actinomycetes, proactimomycetes, micromonospores, protozoa, algae, etc against which no antagonist can be found. In laboratory practice, one usually gives the name antagonist only to a microbe which suppresses the widely used test organisms, which usually comprise a narrow range of known cultures of bacteria or fungi. One does not take in account the fact that the so-called inactive forms (nonantagonists) in these tests might be active against other organisms. On the basis of our own experience and data from literature, we can say that the property of antagonism is characteristic of all species of microorganisms, but is expressed differently and to various degrees depending on the natural properties of the antagonist on the one hand, and the sensitivity of the test organism on the other hand, and also upon the type of substrate and other external conditions.

  The antimicrobial action of the antagonists manifesto itself not only under laboratory conditions on artificial media but also under natural conditions of habitation, in the soil. In sterile soil, where there are no antagonists the growth of microbes and the biochemical processes which they cause take place at an intense rate. However, it is sufficient, to introduce an antagonist in such soil in order to stop or to slow down the growth and biochemical processes of microbe.

  In his experiments, Afrikyan (1951) directed the action of antagonists of the group of sporeforming bacteria: Bac. subtilis and Bac. mesenterics against the organisms: Bac. mycoides and Az. chroococcum. The result are given In Table 110.

Table 110
Intensity of Azotobacter growth as depending on
growth of the antagonistic bacterium--Bac. mesentericus
(experiment with sterile soil; table shows number of cells, in thousands,
in 1 g of soil after passage of days)

Experimental conditions	Initial number	1	2	3	5	7	10
No antagonist (control)	80	150	380	520	760	800	1,000
With antagonist	80	100	30	0.05	0	0	0.05
No antagnist (control)	200	500	600	800	800	800	500
With antagonist	200	100	50	0.5	0	0	0.05
Antagonist	80	400	1,200	1,500	1,600	1,000	800

  Bac. mesentericus was introduced into sterilized soil together with cultures of Azotobacter to which it is an antagonist, The growth of Azotobacter was suppressed by Bac. mesentericus. Under conditions of growth in sterilized soil when grown alone, Azotobacter reached 1 million cells per 1 gram of soil and in the presence of Bac mesentericus, Azotobacter grew very slowly and only during the first day; then the number of its cells decreased rapidly, dropping practically to zero. Only at the end of the experiment, after 10-15 days, when growth of the antagonist and formation of the antibiotic stopped, did the Azotobacter resume growth although at a very slow rate.

  The same was observed in experiments with Bac. mycoides. This microbe in very sensitive to the antibiotic action of the potato bacillus. In an isolated condition, in sterilized soil, its growth is excellent but it ceases to grow completely or almost completely in a mixture with the antagonist (Figure 92).

Figure 92 Growth of Bac. mycoides in soil (sterile) in presence of the antagonist--Bac. mesentericus:

1--growth of Bac. mycoides without antagonist; 2--growth of Bac. mesentericus in soil; 3--growth of Bac. mycoides in a mixture with Bac. mesentericus.

  Mikhaleva (1951) studied the growth of the root-nodule bacteria of clover, peas, kidney, beans, lupine, soy, lucerne, etc in the presence of antagonists and actinomycetes, The most resistant, according to their data, were the soy bacteria In podsol soils these bacteria are more strongly suppressed by actinomycetes than in chernozem soils. Root-nodule bacteria of clover perish after 4 days in the soil in the presence of the antagonist.

  The activity of the root-nodule bacteria decreases markedly under the influence of the antagonist; the number of nodules on the roots is usually smaller than in the controls. According to our observations, the root-nodule bacteria of clover vetch and peas react noticeably to the action of antagonistic Bac. subtilis, Bac. mesentericus and others. When the soil is artificially enriched with these bacteria the nodules do not develop on the roots of the above-mentioned plants or they appear in small numbers only, with a degenerated appearance, an unusual form and a small size.

  Stolp (1952) studied the growth and activity of root-nodule bacteria of peas in the presence of Pennicillium expansum. Robinson (1946) observed cultures of antagonists among actinomycetes, bacteria and fungi, which actively suppressed the growth and virulence of root-nodule bacteria in the laboratory and under field conditions as well.

  The general importance of bacterial antagonists is determined not only by the nature and strength of their activity but also by their number in the soil. The more intensely they grow in the soil, the higher their concentration and the stronger the effect they exert.

  It is difficult or almost impossible to assess the total number of antagonists in the soil. As was already mentioned, all microbes possess antagonistic proprties against some microbe. However, it is practically impossible to detect antagonism against all existing microorganisms.

  In our studies we give quantitative indexes of distribution. of antagonists in relation to certain species of bacteria or fungi. Among the bacteria, cultures of Azotobacter, root-nodule bacteria, mycobacteria, micrococci and nonsporeforming bacteria (colon bacillus, etc) were used.

  According to our calculations, there are very large quantities of actinomycete-antagonists with clearly expressed antimicrobial properties in different soils.

  In the previous chapter (microbial inhibitors) data were presented on the number of microbes which suppress the growth of Azotobacter. The number of bacterial antagonists ranged between 10,000 and 450,000; fungi, between 1,300 and 17,000 and actinomycetes, from 10,000 to 160,000 in 1 g of soil, depending on the properties of the latter.

  In relation to certain other species of bacteria (Bac. mycoides, Bac. subtilis) and fungi (Fusarium lini, Fusarium sp., etc) the total number of antagonists is much higher. In our analyses of various soils of the Soviet Union we found tens and hundreds of thousands and often millions of them in 1 g of soil.

  In the podsol soils of the Moscow area one may find 40,000- 1,000,000 actinomycetes-antagonists or even more, per 1 g of soil; sporeforming bacteria in numbers of 20,000-500,000 may be found in 1 g of soil. In the chernozems of the southern districts of the USSR the total number of microbes is higher and, as a rule, the number of antagonists is also higher. However the percentage of antagonists may be lower. For example, the soils of the Crimean steppes contain a smaller percentage of antagonists than the northern soils of the Kola Peninsula or the soils of the central districts. There are relatively few antagonists in the red soils of the Caucasian coastal area (Table 111).

  In certain soils one may find 2,000-400,000 bacterial antagonists per 1 g of soil; they belong to two groups--Bac. subtilis and Bac. mesentericus. Among the nonsporeforming bacteria and especially among species of the genera Pseudomonas and Bacterium, there are many antagonists. A special place in relation to their quantity among the antagonists of this group is occupied by the mycolytic bacteria. These bacteria grow well in many soils and in the rhizosphere of various plants. According to our figures, their total number approaches tens of millions and more in 1 g of soil (Krasil'nikov, 1940 a,b, c; Kusina, 1951; Kublanovskaya, 1953).

  Rasnitsyna (1947) studied the soils of the Vakhah valley and counted 100,000 to 100 million mycolytic bacteria (which lyse the fungus Fusarium vasinfectum) in 1 g of soil, depending upon the vegetative cover. The greatest number was found in the rhisosphere of lucerne, spear grass and the Euagropyrum; they were less numerous under rye grass, brome grass and peas. Kuzina (1955) gives more or less similar indexes for light serozems of the Uzbek SSR.

  Novogrudskii (1949 a) isolated mycolytic bacteria from Kazakhstan soils which lyse the fungi: Fusarium culmorum, F. graminiarum, Verticillum dahliae, Colietotrichum lini, Alternaria tenuis, Amblyosporium botrytis, Pyronema confluens, and Mucor racemosus.

  In the same soils the author counted from 100 to 100 thousand mycolytic bacteria in 1 g of soil. These are active solely against Fusarium graminiarum and were more numerous under lucerne than under oat or millet.

  Above were given data on growth and accumulation of these bacteria in soils under different plants.

  In nature microorganisms do not live alone, but in associations which contain many foreign species--competitors and noncompetitors. In these associations definite and quite complicated relations are established among the species, of both a symbiotic and an antagonistic nature.

  In microbial populations or coenoses, each species in its struggle for survival throughout a long history of evolution, elaborated certain means of struggle with its competition. These means are versatile. Microbes may displace their competitors by abundant multiplication or they may form during their metabolism various specific and nonspecific substances which suppress microbial growth. These nonspecific substances are, for example, organic acids, alcohols, peroxides and other compounds. These metabolic products are characteristic of many microbial species.

  Examples of the nonspecific metabolites are certain inorganic substances, such as ammonia, hydrogen sulfide, ions of chlorine, SO₃, aluminum, iron and other elements, Plentiful emission of H₂S, formed as a result of the vigorous metabolic activity of the appropriate microbes, may cause the poisoning of the milieu, making it inadequate for the growth of many foreign species of microbes and even for higher plants, as was observed by us in soils with high ground-water level in the Trans-Volga region (Krasil'nikov, Rybalkina and others, 1934).

  Microbes producing acids, alcohols and other organic compounds suppress the growth of organisms which are sensitive to these substances, regardless of the species to which they belong.

  The most characteristic and outstanding reactions are those which are caused by particular specific substances, the so-called antibiotics, which act against microbes. These substances have specific effects. The microbial antagonists which form these substances suppress growth of certain specific species only. Some antagonists inhibit only gram-positive bacteria and others, inhibit both grarn-positive and gram-negative bacteria. Some of them act on cocci and others on bacilli.

  Some antagonists have an inhibitory effect only on fungi or only on phages and viruses, etc.

  Antibiotic substances produced by antagonists are a potent weapon in the struggle with competing microbes.

  A characteristic feature of such antagonists is the fact that as a rule, they act only upon foreign species. A. streptomycini, the producer of streptomycin, does not inhibit cultures of its own species. The producer of aureomycin, A. aureofaciens does not inhibit cultures of its own species, regardless of from where they were isolated or under what conditions they lived previously. Similarly, other antagonists which produce antibiotic substances, terramycin, chloromycetin, actinomycin, sulfactin, etc do not suppress the growth and development of strains which belong to the same species.

  These characteristics of specific or selective action of antagonists determine to a large extent the species makeup of microbial populations in natural substrates.

  Experience shows, that there are microorganisms that form antibiotics under conditions of solitary growth, on artificial nutrient media. Their ability to form these antibiotic substances is hereditarily fixed and expresses itself in the absence of competitors.

  These organisms are looked upon as potent antagonists. They are often found among various microbial species. They comprise the main group of producers of antibiotic substances obtained to date in various laboratories and in the antibiotic industry. In other species this property is not fixed by heredity and appears only in the presence of competitors, i.e., under conditions of mixed populations. In pure isolated cultures these organisms do not produce antibiotic substances. For example, the colon bacillus, Bact. coli suppresses the growth of Bac. anthracis only in those cases where both organisms are together. In a pure culture the colon bacillus does not produce antibiotic substances which are active against the mentioned microbe. Apparently these microbes only produce antibiotic substances under pressure, out of necessity.

  Shiller in 1914 was the first to pay attention to the forced nature of the formation of antibiotics by microorganisms. He was working in Mechnikov's laboratory on the interrelations between the acidophilic bacilli, sporeforming bacteria, streptococci, pneumococci and other species (Shiller, 1952).

  The phenomenon of forced antagonism was also noticed by other investigators (Peretts and Slavskaya, 1934; Izabelinskii and Soboleva, 1934 and others). Streshinskii (1949, 1950) has demonstrated the formation of antibiotic substances in mixed cultures of a fungus and a sporeforming bacillus. According to his observations, Bac. subtilis forms an antibiotic substance against the Penicillium fungus only when the two grow together. The fungus too becomes a more active antagonist in the presence of the bacillus. We observed forced antagonism in a number of actinomycetes. Cultures grown separately on nutrient media, do not form antibiotic substances, but in the presence of certain microbes (fungi or bacteria) these substances which suppress the growth of competitors are formed. Two inactive species of actinomycetes when grown together, form antimicrobial substances.

  All such organisms possess a latent antagonistic capacity which is not fixed hereditarily.

  The majority of microbial antagonists readily lose their ability to produce antibiotics, active strains turning inactive in the process of variation. This property is encountered in many organisms used in industry and causes many difficulties in the antibiotic industry and in laboratory practice.

  Antibiotic substances should be classified an potent weapons in the struggle of microbes with neighboring competitors; as a biologically important attribute formed in complex populations during phylogenesis and as a property determining the degree of development and distribution of the microorganism in nature. The biological role of antibiotic substances and, therefore, the phenomenon of antagonism as a whole, is quite important in the life of both higher and lower soil organisms.

  Through use of their metabolic products antagonistic microbes suppress their competitors, removing them from the substrate and thus exerting a definite selective action. To a certain degree, microbial antagonists regulate the formation of microbial coenoses (colonies) in the soil in general. They play an important role in the improvement of soils, in the so-called process of self-purification of soils. The removal of harmful pathogenic and phytopathogenic flora and fauna is accomplished by microbial antagonists.

  Microbial antagonists suppress not only the growth and propagation of competing organisms, but also many of the functions of the latter. There are among microorganisms those which inhibit certain processes which occur in microbes.

  Inhibitors were observed to suppress nitrification, denitrification, nitrogen fixation, decomposition of cellulose, etc. Appropriate cultures inhibit the decomposition of organic substances, fermentation of sugars, etc. Microbial cultures are known which inhibit the formation of certain metabolites, various acids, enzyme biotic substances, auxins, vitamins, amino acids and other biocatalysts. There are organisms in the soil which, with their metabolic products, suppress the formation of toxins and antibiotics and often inactivate them in the medium, if they are formed there.

  There are data in the literature dealing with the suppression by inhibitors and their metabolites of processes of cell multiplication, spore formation, budding and the sexual process. Inhibitors often inhibit the process of respiration.

  In the presence of antagonists or their metabolic products many active substances: biocatalysts, antibiotics, toxins, and enzymes lose their peculiar functions, and become inactive. Toxins become harmless, antibiotics no longer suppress the corresponding microbes, enzymes no longer cause the decomposition of organic matter, etc. In general, one may say that for any metabolite, living nature creates an antimetabolite (Woolley, 1954). The metabolite penicillin becomes nonbactericidal for staphylococci and certain other bacteria, which produce an antimetabolite penicillinase (Abraham and Chain, 1940; Woodruff and Foster, 1945). Sulfonamide compounds are inactivated by bacteria which produce paraaminobenzoic acid (Woods, 1940; Sevag and Green, 1944, 1950) or the factors H.P. etc (Moller and Schuerz. 1941; Green 1940). Mucin suppresses the antibacterial action of tyrothricin; tannic acid neutralizes actinomycin (Waksman, 1947). In our bacterial collection there were strains, which completely inactivated the antibiotic substances, formed by the sporeforming bacteria: Bac. subtilis, Bac. mesentericus and Bac. cereus. We also possessed bacterial cultures, which annuled the toxic effect of Ps. pyocyanea metabolites. The toxin of the fungus Botrytis cinerea is neutralized by metabolites of certain actinomycetes.

  A solution of mycetin at a 1:10,000 dilution completely suppresses the growth of Staph. aureus and in the presence of filtrate or cells of a Bac. proteus culture the antibacterial effect is either nonexistant, or becomes very weak. A culture of the colon bacillus and certain Pseudomonas strains had such an inactivating effect in our experiments. An inactivating effect of microbes was also found in relation to other antibiotics (Krasil'nikov and Nikitina, 1951).

  As seen from the above-mentioned data, antagonists exert a definite suppressing effect on various microorganisms. Due to their action, they have a considerable influence on the formation of microbial coenoses in soil in general and determine, to a certain degree, the distribution and accumulation of the various species of soil microflora.

  Antagonists, therefore, can be considered as one of the powerful factors governing soil fertility and plant-crop abundance.

  It is only natural that this group of microorganisms attracts the attention of specialists in many fields--microbiologists, phytopathologists, plant breeders and others. The possibility of practical utilization of antagonists is one of the most important reasons for the study of the phenomenon of antagonism.

  As was noted before, in soils in which antagonists grow abundantly (bacteria, fungi or actinomycetes). microbes, sensitive to them, saprophytes as well as phytopathogens, grow much more slowly, or not at all. This served as a basis for the use of microbial antagonists in the struggle against harmful microflora, and against organisms causing plant disease.

  The first attempts in this direction were made by Porter (1924). He treated wheat seeds with bacterial antagonists and then infected them with Helminthosporium fungi. The seeds either did not become infected and germinated normally, or they were slightly affected. Bamberg (1931) infected wheat seeds in order to protect them against smut. Weindling (1940, 1948) used the fungus Trichoderma lignorum for the protection of citrus saplings from Rhizoctonia. This fungus according to other authors, also protects cucumbers and peas from Rhizoctonia and wheat from fusariosis (Allen and Haenseler, 1935; Bisbu, James and Timonin, 1933). Milliard and Taylor (1927) observed a protective effect of the actinomycetes-antagonist---A. praecox in relation to the agent of scab in potatoes A. scabies. No scab was observed upon prolific growth of the antagonist.

  A similar phenomenon was described by Kissling (1933). He experimented with bacterial antagonists in subduing potato scab. Under conditions of a field experiment, upon sufficient growth of the antagonist the disease either did not appear at all or had a limited occurrence only.

  Gregory, Allen, Riker and Peterson (1952) demonstrated the protective role of actinomycete-antagonists in their experiments with the phytopathogenic fungi Pythium ultimum and Pythium debaryanum. These fungi were quite virulent for lucerne. The introduction of certain species of actinomycotes-antagonists into the soil (Actinomyces sp.) protected the plants from the disease. A positive result was also obtained through use of antagonists--fungi Trichoderma lignorum and Penicillium patulum, and the sporeforming bacteria, Bacillus sp. No 6.

  Rehm (1953) treated wheat and rye seeds with actinomycetes-antagonists in order to fight the organisms which cause fusarlosis (Fusarium nivale and Fus. culmorum). As a result, seed germination Increased by 30 %.

  Gorrard and Lockhead (1938) compiled a long list of microbial antagonists which suppress the development of phytopathogenic organisms in soil. Among them there are sporeforming and nonsporeforming bacteria, fungi, actinomycetes and protozoa. Cordon and Haenseler (1939) have shown experimentally the inhibitory effect of the sporeforming Bacillus simplex on the growth and development of the fungus Rhizoctonia solani which affects cucumbers and peas. A culture of the antagonist was introduced Into the soil in which the plants were grown. As a result, the morbidity of the latter dropped. In the control without bacterial antagonists 65 % of the total number of cucumber plants and 48% of peas were affected; after introduction of the antagonist the mortality role was 35 and 45% respectively.

  Ark and Hunt (1941) mention two cultures of sporeforming bacteria--Bac. vulgatus and Bacillus sp. which suppressed the growth of many phytopathogenic bacteria in soil and protected the plants from disease. Other investigators also speak of the antagonistic action of microbes in soil against phytopathogenic bacteria and fungi (Johnson, 1935; Eaton and Rigler, 1946; Allen and Haenseler, 1935; Feeney and Garibaldi, 1948; Kenknight, 1941 and others).

  In the Soviet Union, as already mentioned above, Khudyakov (1935) and Novogrudskii (1936) established the lytic effect of mycolytic bacteria on phytophatogenic fungi. These bacteria were subsequently exclusively tested fighting plant infections under laboratory conditions, and in open fields as well. The results were positive in many cases.

  The percentage of mortality noticeably decreased. For example, the experiments of Berezova (1939), performed on kolkhoz fields with flax, have shown, that mycolytic bacteria lower the incidence of fusariosis by an average of 40% and in isolated cases their action was even more effective.

  Raznitsyna (1942) used mycolytic bacteria which are isolated from the soil against fusariosis of pine saplings (Figure 93). Under open-field conditions on a sector strongly affected by fusariosis, inoculation of bacteria gave an outright positive effect, and the lowering of morbidity reached 80 % and more (Table 112). The plants looked considerably healthier on sectors treated with mycolytic bacteria, than in the control. The needles were longer, stronger and greener, the stem of the saplings thicker and taller than in saplings not treated with bacteria (Figure 94).

Figure 93. Protective action of bacteria in fusariosis of pine saplings:

1--plants infected with the fungus Fusarium; 2--plants also infected with Fusarium, but treated with mycolytic bacteria.

Figure 94. Pine saplings growing on sectors affected by fusariosis:

a--not treated with bacteria; b and c--treated with mycolytic bacteria.

  Korenyako (1940) studied mycolytic bacteria in the soils of the Uzbek SSR for a number of years. She isolated cultures of bacteria of the genus Pseudomonas, which showed antagonistic properties against the agents causing wilt of the cotton plant Verticillium dahliae. When these bacteria were tested on the experimental fields of STAZRA SOYUZNIKHI (Tashkent) which were strongly affected by the mentioned fungus, positive results were achieved. The mycolytic bacteria suppressed the growth of the fungus and lowered the morbidity of the cotton plant by 60- 80%. Kuzina (1951) and Kublanovskaya (1953) corroborated these results. They showed that mycolytic bacteria in mixtures with other bacteria suppress phytopathogenic fungi more vigorously.

  Davydov (1951 used mycolytic bacteria against the mildew organism (Sphaerotheca mors uvae) on the gooseberry shrub. The parasitic fungus disappeared upon introduction of the bacterial antagonists and the plant recovered and developed normally.

  Petrusheva (1953) used cultures of actinomycetes as antagonists against tobacco-seedling rot caused by the fungus Thielaviopsis basicola, and against blackleg of citrus cultures, caused by Fusarium sp. In soil treated with antagonists the plants grew normally; when the actinomycetes were not introduced into the soil, the mortality of the plants reached 70%.

  Gurinovich (1953) used cultures of actinomycetes and bacterial antagonists in the struggle against the vascular disease of cabbage caused by nonsporeforming bacteria Pseudomonas campestris. The antagonists were introduced into the soil affected by the soil microbe, and prevented the plants from becoming infected.

  Seiketov (1951) tested four cultures of the fungus Trichoderma--T. glaucum T. lignorum, T. koningii and T. album as antagonists of Rhizoctonia, which affects potatoes. The percentage of mortality of the "early rose" variety was considerably lower (7-8%) in the experimental plants than in the controls (54 %).

  Kuzina (1955) used mycolytic bacteria against wilt of the cotton plant caused by verticillium. She treated the seeds with bacteria before sowing. The morbidity in the control sectors was 54% and after treatment with the bacterial antagonists it was 8--9%. Correspondingly, the crop yield was: in the control- 1, 030 bolls and the total weight of seed cotton was 342 g. In the experimental plants there were 1,630 bolls and the weight of the seed cotton was 514 g, i.e., 57% higher than those of the control.

  Kublanovskaya (1953) used actinomycetes as antagonists to fight cotton-plant wilt. She prepared a special composted preparation of cotton cake with actinomycetes. This was introduced into the soil in which the cotton seeds were sown. The actinomycetes introduced in this manner proliferated abundantly in the soil and suppressed the growth and activity of the fungi Verticillium dahliae and Fusarium vasinfectum, protecting the plants from the disease. At the end of the growth period the number of plants in the cotton plants of the 8517 variety affected by the wilt was: 52.6% of all control plants and 18.1% in sections treated with actinomycetes; in the cotton plants of the 108-F variety there were 23.3 % diseased control plants and 3.3 % treated plants. In the treated sections all the plants looked stronger, the bushes were larger, the leaves wider and flowering was more abundant, etc (Figure 95).

Figure 95. Protective effect of actinomycetes-antagonists against cotton-plant wilt caused by Verticillium:

a) plants affected by wilt, not treated with the actinomycete: b) plants whose seeds were treated with a culture of actinomycetes (according to Kublanovskaya (1953).

  The crop in these experiments also increased noticeably in sectors treated with the antagonists. The 8517 variety yielded: 9.4 centners/ha in the control sector and 22.6 centners/ha in the experiment. The variety 108-F yielded: 17.8 centners/ha in the control sector and in the experiment 22.8 centners/ha.

  Chinese scientists Yin, Chen, Yang et al., (1955) corroborated Kublanovskaya's data. They prepared compost from soil with cotton cake and grew actinomycetes in it which were antagonists of the wilt fungus Verticillium dahliae. The ripened compost was used for the treatment of the seeds. The latter were sown together with the compost. The incidence of the wilt disease decreased by 50-75 %, the cotton crop increased by 13-45%.

  Mitchell et al, (1948) observed vigorous growth of antagonists-actinomycetes and bacteria, when those were introduced in the soil together with a composted plant mass. The phytopathogenic fungus causing root rot in the cotton plant perished. The positive role of composted preparations saturated with microbial antagonists was noted by Sanford (1946-1948). He observed in his experiments a drop in the morbidity of potato tubers, pine saplings, etc. Similar data are given by some other investigators (Weindling, 1946; Garret, 1946; Winter and Rümker, 1950). They all noted that the favorable action of composted preparations was not due to an improvement in plant nutrition but to the more intense development of antagonists in the soil.

  Schaffnit and Neuman (1953) used peat composts, enriched with bacterial antagonists on the snowy mold caused by Fusarium nivale. The preparations were made from various types of post. The best results were achieved with bog peat of mass origin. The percentage of morbidity in wheat treated with composted peat was considerably lower than in the control.

  Wood and Tveit (1955), on the basis of data from the literature and their own observations, came to the conclusion that microbial antagonists play an important role in soil improvement. Microbes of local origin are much more effective. In order to enhance their growth and activity the authors suggest the introduction of certain plant residues into the soil as sources of nutrition.

  There are many other studies in the literature which confirm the positive effect of microbial antagonists in the struggle against phytopathogenic fungi, bacteria and actinomycetes. All these studies show that microbial antagonists can be used in agricultural practice for the improvement of soils. For this purpose the soil should be enriched with the appropriate antagonists.

  This can be achieved by various methods. As may be seen from the above data the antagonists mentioned may be introduced into the soil in the form of pure cultures (which is not very effective), or in the form of composts.

  In the enrichment of soils with antagonists, the vegetative cover plays an especially important role. It was noted above that the plants are a powerful selective factor. Some of them, under certain conditions favor the growth and accumulation of phytopathogenic microbes in the soil, and others favor the growth of the antagonists of these microbes. By selecting, by means of special experiments, those plants in whose rhizosphere the needed antagonists grow abundantly, and by using these plants in the crop rotation, one may remove or suppress the growth and the harmful activity of the pathogenic microbe.

  If the selected plants are inoculated with microbial antagonists before sowing, their accumulation in the soil can thus be markedly enhanced.

  Upon introduction of pure cultures of antagonists, one must take into account their adaptability, their growth in the soil, and their activity. If the antagonists lose their activity in the soil or in a substrate which is not suitable for them which often happens), or if they do not grow or grow but little, their effect will be small or will not express itself at all.

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